1. Field of the Invention
Infection by Neospora parasites results in neosporosis, a disease which causes paralysis and death in dogs (Dubey et al. 1988. J. Am. Vet. Med. Assoc. vol. 192, pp. 1269-1285) and abortion and neonatal morbidity and mortality in cattle, sheep, goats and horses (Dubey and Lindsay. 1993. Parasitol. Today. vol. 9, pp. 452-458). Methods for detection of the parasite are available, however, definitive diagnosis of neosporosis has often been difficult to achieve. This invention relates to recombinant antigens isolated from Neospora caninum (N. caninum) which have provided an effective means for diagnosing the disease.
2. Description of the Related Art
Diagnosis of abortion due to neosporosis may be achieved by demonstration of the presence of the parasite in tissues of aborted fetuses by immunohistochemical identification (Lindsay and Dubey. 1989. Am. J. of Vet. Res. vol. 50, pp. 1981-1983). Few parasites are present, however, and aborted fetuses may be severely autolysed; therefore, recovery of parasites from fetal samples useful for this purpose is often not possible.
An immunofluorescent antibody test (IFA) has been developed for the serodiagnosis of neosporosis using N. caninum tachyzoites. The use of whole tachyzoites for immunodiagnosis may result in false positives, however, since there appear to be antigens that are conserved between N. caninum tachyzoites and the closely related protozoan Toxoplasma gondii (Conrad et al. 1993. J. Vet. Diagn. Invest. vol. 5, pp. 572-578; Dubey and Lindsay, supra), therefore some cross-reaction may be expected to occur. Other drawbacks associated with the tachyzoite IFA include the requirement for growing the parasite in vitro, which is time-consuming and expensive, and the need for trained personnel to interpret IFA slides.
A method for the detection of antibodies to N. caninum in serum from dogs by indirect enzyme-linked immunosorbent assay (ELISA) has been described (Bjxc3x6rkman et al. 1994. Parasite Immunology. vol. 16, pp. 643-648). This method utilizes proteins extracted from N. caninum tachyzoites as antigens. Similarly, an ELISA for the diagnosis of neosporosis in cattle was developed using sonicated tachyzoites of Neospora isolated from an aborted bovine fetus as antigen (Parxc3xa9 et al. 1995. J. Vet. Diaan. Invest. vol. 7, pp. 352-359). These tests are capable of detecting the presence of antibodies to Neospora sp.; however, as with the IFA method, antigens which are conserved between Neospora sp. and other similar protozoa are more than likely present in the antigen preparations, thus the possibility of cross-reaction still exists. The search therefore continued for antigens having increased sensitivity and specificity for use in immunological procedures for the diagnosis of the disease.
We have discovered antigens derived from N. caninum tachyzoites which are highly specific and sensitive for the detection of antibodies present in animals suffering from neosporosis and which are not present in morphologically-related parasites.
In accordance with this discovery, it is an object of the invention to provide novel antigens effective for the detection of antibodies to Neospora and for the serodiagnosis of neosporosis.
It is a further object of the invention to provide DNA sequences which encode antigens effective for the detection of antibodies to Neospora and for the diagnosis of neosporosis.
It is also an object of the invention to provide an immunoassay method effective for detecting antibodies to Neospora present in a sample suspected of containing said antibodies.
Other objects and advantages of the invention will become readily apparent from the following description.